The overall goals of this proposal are to improve understanding of the structural basis for the binding and activity of biologically important transition metal ions in metalloproteins, to test this understanding by engineering, characterizing, and determining x-ray structures for site- directed mutant proteins that incorporate transplanted metal-binding sites, and to produce designed metalloproteins that mimic or extend the metal-binding and catalytic properties of the original metal-binding site. We will use recursive structure-based design to transplant metal- ions templates from Cu- and Zn-containing metalloproteins of known structure into three different protein structural frameworks. For protein scaffolds, we will start from the known, high resolution, crystallographic structures of antibodies, photoactive yellow protein (PYP) and green fluorescent protein (GFP). By using the same metal ion templates with different protein scaffolds and among different locations within a single scaffold, we will test the roles of the protein framework in determining metal-site geometry and providing the environment needed for the affinity, specificity, and activity of metal- binding sites in proteins. After design and construction, biochemical and spectroscopic characterization, and 3-dimensional structure determination and analysis of these metalloprotein mutants, we will apply our results and those from other projects to improve and optimize the original designs, or to suggest new designs. The results from the other projects on the structural and chemical roles of metal sites in the folding and assembly, stability, regulation, and chemical activity of metalloproteins will contribute experimental and computational information to improve our metalloprotein designs. This interactive approach will allow us to rigorously test and refine our understanding of the requirements for functional metal sites in proteins. The long term objective of this proposal is to engineer scientifically and medically useful metalloproteins that synergistically combined the specificity of antibodies or the natural reporter groups of PYP and GRP, with the remarkable functional and catalytic properties of protein-bound metal ions.